4-Hydroxytamoxifen (4-OHT), a metabolite of tamoxifen (TAM), the most widely used antiestrogen for the treatment of advanced breast cancer, binds the estrogen receptor (ER) with high affinity. 4-OHT-ER binds to specific DNA sequences called estrogen response elements (EREs), located near or in the coding regions of estrogen-regulated genes with an affinity comparable to estradiol (E2)-liganded ER. This interaction initiates a series of events that alter gene transcription. Differences in binding of E2-ER versus 4-OHT-ER to EREs were revealed by use of single and multiple tandem ERE constructs that were varied in composition, spacing and helical orientation. Of note was our finding that dissociation of one 4-OHT ligand molecule occurs when dimeric 4-OHT-ER binds to EREs. Similarly, EREs lacking an AT-rich flanking sequence promoted dissociation of one E2 ligand molecule from the E2-ER dimer. Thus, the composition of flanking regions facilitates conformational alterations in the ER that impact ligand binding stability and induction of responsive genes. Although ER does not bind an ERE half site, we discovered a protein that binds a half site ERE and that interacts with ER in a ligand-specific fashion. We will address the mechanisms behind these observations, using equilibrium ERE binding, gel shift, footprinting, and transfection assays to: 1) define the determinants and mechanism by which AT-rich sequences in the ERE flanking region and the length of the ERE inverted repeat influence ER conformation and ligand stability; 2) establish what structural features of tandem EREs account for functional synergy of reporter gene transactivation in vivo; 3) ascertain whether the dissociation of one 4-OHT ligand molecule from the ER is a more general feature of antiestrogen action by studying the ERE binding properties of raloxifene-liganded ER; and 4) determine the role of a newly identified ER-associated DNA binding protein on 4OHT- and E2-ER-ERE binding and on estrogen-regulated gene transcription. Results will define how antiestrogens, and the ERE and its flanking sequence, impact ER conformation, ERE binding affinity, and the role of an auxiliary protein on induction of gene expression.